The long-term goals of this proposal are to examine the structure, function, and mechanism of action for a set of enzymes required for the biosynthesis and catabolism of mammalian N-linked glycans. We have focused on the Class I (family 47) a-mannosidases in the N-glycan pathway since they act as committed steps in the synthesis of complex oligosaccharides by determining the extent of mannose trimming, and in some cases appear to control the rate of degradation of unfolded glycoproteins in the endoplasmic reticulum (ER). Three subfamilies of Class I mannosidases have emerged from our prior cloning studies. The ER mannosidase I subfamily cleaves a single residue from Man9GlcNAc2 to generate a specific Man8GlcNAc2 isomer. The Golgi mannosidase I subfamily cleaves Man9-8GlcNAc2 structures to Man5GlcNAc2. The third subfamily, termed EDEM for ER degradation enhancing a-mannosidase-like protein, do not appear to have an intrinsic hydrolase activity, but may have a lectin activity involved in glycoprotein degradation. Recently, both ER Man I and EDEM proteins have been implicated as key players in the targeting of unfolded glycoproteins for disposal in the ER. This application will examine the mechanism used by the Class 1 mannosidases to recognize unfolded glycoproteins for disposal with a goal of understanding how intervention in the process can lead to enhanced protein stability in human genetic diseases characterized by the rapid degradation of unfolded mutant glycoproteins. Four specific aims are addressed in this proposal. The first aim will test hypotheses for the novel hydrolase mechanism and specificity of binding by Class 1 mannosidases by a combination of mutagenesis, kinetic analysis, binding studies, and structural analysis to map the determinants of substrate recognition and catalysis among the hydrolase family members. The second aim will test hypotheses relating to the lectin binding specificity of EDEM subfamily members through in vitro binding studies using wild type and mutant forms of recombinant EDEM or EDEM sub-domains in combination with in vivo functional complementation in model ER-associated degradation (ERAD) systems. The third aim will test hypotheses relating to EDEM function by examining sequences necessary for localization and interactions with components of the ERAD machinery. The fourth aim will test hypotheses for the individual roles and mechanisms of regulation of members of the ERAD targeting machinery by a novel real-time RT-PCR strategy and by siRNA approaches.